Deuterated bile acids

ABSTRACT

This disclosure relates to deuterated bile acid compositions. A deuterated compound is selected from the disclosed groups of bile acids and their derivatives, analogs and salts. At least one of the hydrogen atoms in the compound is replaced with deuterium.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. application Ser. No.14/520,889, filed Oct. 22, 2014, and entitled “DEUTERATED BILE ACIDS”,which claims priority from U.S. Provisional Application Ser. No.61/894,012, filed Oct. 22, 2013, and entitled “DEUTERATED BILE ACIDS”.

BACKGROUND

Ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) areanti-apoptotic molecules with protective effects against severalneurodegenerative disorders such as Alzheimer's and Parkinson's diseasesas well as against acute kidney injury. Both UDCA and TUDCA block theinitiating event of the apoptotic process, in part, through stabilizingthe mitochondrial membrane potential, a mechanism that enhances theintegrity of the mitochondria. Enhanced mitochondrial integrityabolishes the release of several mitochondrial proteins such ascytochrome C into the cytosol, thereby preventing the onset ofapoptosis. Further, UDCA and TUDCA upregulate several pathways thatfunction synergistically with their anti-apoptotic properties.

The kinetic deuterium isotope effect (KDIE) is a function of enhancedcarbon-deuterium (C-D) bond strength over the carbon-hydrogen (C—H)bond, often several-fold. Substitution of the C—H by the C-D bondsignificantly decreases breakage, thereby increasing the resident timeof the molecule in the body. Therefore, substituting one or more of thecarbons with deuterium significantly increases the metabolic clearancetime. An added benefit of the KDIE is the possibility of using lowerdosages to derive the same pharmacological effect. The C-D bond is twiceas strong as the C—H bond by virtue of a two-fold higher mass ofdeuterium over hydrogen. Hence, the reaction rate of the C-D bondbreakage is significantly slower than that of the C—H bond breakage.

SUMMARY

This disclosure relates to deuterated bile acid compositions. Adeuterated compound is selected from the disclosed groups of bile acidsand their derivatives, analogs and salts. At least one of the hydrogenatoms in the compound is replaced with deuterium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structure of UDCA.

FIG. 2 illustrates the structure of TUDCA.

FIG. 3 illustrates the structure of lithocholic acid (LCA).

FIG. 4 illustrates the structure of dehydro(11,12)ursolic acid lactone.

FIG. 5 illustrates the structure of ursolic acid.

FIG. 6 illustrates the structure of ursocholanic acid.

FIG. 7 illustrates the effect of deuterated UDCA on DCA-inducedcytotoxicity and apoptosis in primary rat hepatocytes.

FIG. 8 illustrates the effect of deuterated UDCA on TGF-β1-inducedcytotoxicity and apoptosis in primary rat hepatocytes.

DETAILED DESCRIPTION

This patent application pertains to the field of pharmaceuticalmolecules and specifically to deuterated versions of bile acids, such asUDCA and TUDCA as well as their analogs and derivatives, with enhancedresident time following administration to a patient.

Deuteration of bile acids can significantly increase the half-life ofthe drug in the bloodstream and, hence, decrease the dosage needed totreat various degenerative disorders. Disclosed herein are variouscompositions of deuterated bile acids and their analogs and derivatives,as well as methods of preparation.

In some embodiments, UDCA, its analogs and salts have the structure ofFormula I as illustrated in FIG. 1 and reproduced below:

where R₁-R₄₀ are individual hydrogen or deuterium atoms. Any analog orsalt of UDCA can contain at least one deuterium atom represented by anyof the R₁-R₄₀ locations in any combination.

In another embodiment of formula I, the deuterated UDCA, UDCA analog orUDCA salt contains one or more PO₄ groups, preferably in positions 3, 7,24.

In other embodiments, TUDCA, its analogs and salts have the structure ofFormula II as illustrated in FIG. 2 and reproduced below:

where R₁-R₄₄ are individual hydrogen or deuterium atoms. Any analog orsalt of TUDCA can contain at least one deuterium atom represented by anyof the R₁-R₄₄ locations in any combination.

In another embodiment of Formula II, the deuterated TUDCA, TUDCA analogor TUDCA salt contains one or more PO₄ groups, preferably in positions3, 7, 24.

In other embodiments, lithocholic acid (LCA), its salts, derivatives andanalogs have the structure of Formula III as illustrated in FIG. 3 andreproduced below:

where R₁-R₃₉ are individual hydrogen or deuterium atoms. Any analog orsalt of LCA can contain at least one deuterium atom represented by anyof the R₁-R₃₉ locations in any combination.

In another embodiment of Formula III, the deuterated LCA, LCA analog,LCA derivative or LCA salt contains one or more PO₄ groups.

In other embodiments, dehydro-(11,12)-ursolic acid lactone, its salts,derivatives and analogs have the structure of Formula IV as illustratedin FIG. 4 and reproduced below:

where R₁-R₄₈ are individual hydrogen or deuterium atoms. Any analog,derivative or salt of dehydro-(11,12)-ursolic acid lactone can containat least one deuterium atom represented by any of the R₁-R₄₈ locationsin any combination.

In another embodiment of Formula IV, the analog, derivative or salt ofdehydro-(11,12)-ursolic acid lactone contains one or more PO₄ groups.

In other embodiments, ursolic acid, its salts, derivatives and analogshave the structure of Formula V as illustrated in FIG. 5 and reproducedbelow:

where R₁-R₅₀ are individual hydrogen or deuterium atoms. Any analog,derivative or salt of ursolic acid can contain at least one deuteriumatom represented by any of the R₁-R₅₀ locations in any combination.

In another embodiment of Formula V, the analog, derivative or salt ofursolic acid contains one or more PO₄ groups.

In other embodiments, ursocholanic acid, its salts, derivatives andanalogs have the structure of Formula VI as illustrated in FIG. 6 andreproduced below:

where R₁-R₅₀ are individual hydrogen or deuterium atoms. Any analog,derivative or salt of ursocholanic acid can contain at least onedeuterium atom represented by any of the R₁-R₅₀ locations in anycombination.

In another embodiment of Formula VI, the analog, derivative or salt ofursocholanic acid contains one or more PO₄ groups.

In another embodiment, structures with formulae I, II, III, IV, V, andVI, and all derivatives thereof are conjugated to anyanti-neurodegenerative pro-drug molecules involved in modulatingneuronal apoptosis.

In another embodiment, structures with formulae I, II, III, IV, V, andVI, and all derivatives thereof are conjugated to pro-drugs ofdopaminergic neurons (DA) neurons such as L-DOPA((S)-2-Amino-3-(3,4-dihydroxyphenyl)propanoic acid) and any analog ofL-DOPA.

In another embodiment, structures with formulae I, II and III and allderivatives thereof are conjugated to glutamate receptor antagonists.

In another embodiment, structures with formulae I, II, III, IV, V, andVI, and all derivatives thereof are conjugated to antioxidants.

In another embodiment, structures with formulae I, II, III, IV, V, andVI, and all derivatives thereof are combined, without conjugation, toany anti-neurodegenerative pro-drug molecules involved in modulatingneuronal apoptosis.

In another embodiment, structures with formulae I, II, III, IV, V, andVI, and derivatives thereof are combined, without conjugation, to anyanti-neurodegenerative pro-drugs of DA neurons such as L-DOPA and anyanalog of L-DOPA.

In another embodiment, structures with formulae I, II, III, IV, V, andVI, and derivatives thereof are combined, without conjugation, toglutamate receptor antagonists.

In another embodiment, structures with formulae I, II, III, IV, V, andVI, and derivatives thereof are combined, without conjugation, toantioxidants.

Typically, for some embodiments, the compound described herein can beformulated in pharmaceutical compositions. A pharmaceutical compositioncontaining a compound of the present disclosure can be administered to asubject, typically a mammal such as a human subject, in a variety offorms adapted to the chosen route of administration. The formulationsinclude those suitable for in vitro cell culture as well as oral,rectal, vaginal, topical, nasal, ophthalmic, parenteral (includingsubcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal,intraventricular, direct injection into brain tissue, etc.)administration.

The formulations can be conveniently presented in unit dosage form andcan be prepared by any of the methods well known in the art of pharmacy.Typically, such methods include the step of bringing the active compoundinto association with a carrier, which can include one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into a desired formulation.

Formulations of the present disclosure suitable for oral administrationcan be presented as discrete units such as tablets, troches, capsules,lozenges, wafers, or cachets, each containing a predetermined amount ofthe described apoptosis-limiting compound as a powder, in granular form,incorporated within liposomes, or as a solution or suspension in anaqueous liquid or non-aqueous liquid such as a syrup, an elixir, anemulsion, or a draught.

The tablets, troches, pills, capsules, and the like can also contain oneor more of the following: a binder such as gum tragacanth, acacia, cornstarch, or gelatin; an excipient such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acid,and the like; a lubricant such as magnesium stearate; a sweetening agentsuch as sucrose, fructose, lactose, or aspartame; and a natural orartificial flavoring agent. When the unit dosage form is a capsule, itcan further contain a liquid carrier, such as a vegetable oil or apolyethylene glycol. Various other materials can be present as coatingsor to otherwise modify the physical form of the solid unit dosage form.For instance, tablets, pills, or capsules can be coated with gelatin,wax, shellac, sugar, and the like. A syrup or elixir can contain one ormore of a sweetening agent, a preservative such as methyl- orpropylparaben, an agent to retard crystallization of the sugar, an agentto increase the solubility of any other ingredient, such as a polyhydricalcohol, for example glycerol or sorbitol, a dye, and flavoring agent.The material used in preparing any unit dosage form is substantiallynontoxic in the amounts employed. The compound can be incorporated intosustained-release preparations and devices, if desired.

A compound suitable for use in the methods of the disclosure can also beincorporated directly into the food of a subject's diet, as an additive,supplement, or the like. Thus, the disclosure further provides a foodproduct. Any food can be suitable for this purpose, although processedfoods already in use as sources of nutritional supplementation orfortification, such as breads, cereals, milk, and the like, areconvenient to use for this purpose.

Formulations suitable for parenteral administration conveniently includea sterile aqueous preparation of the desired compound, or dispersions ofsterile powders having the desired compound, which are preferablyisotonic with the blood of the subject. Isotonic agents that can beincluded in the liquid preparation include sugars, buffers, and saltssuch as sodium chloride. Solutions of the desired compound can beprepared in water, optionally mixed with a nontoxic surfactant.Dispersions of the desired compound can be prepared in water, ethanol, apolyol (such as glycerol, propylene glycol, liquid polyethylene glycols,and the like), vegetable oils, glycerol esters, and mixtures thereof.The ultimate dosage form is sterile, fluid, and stable under theconditions of manufacture and storage. The necessary fluidity can beachieved, for example, by using liposomes, by employing the appropriateparticle size in the case of dispersions, or by using surfactants.Sterilization of a liquid preparation can be achieved by any convenientmethod that preserves the bioactivity of the desired compound,preferably by filter sterilization. Preferred methods for preparingpowders include vacuum drying and freeze drying of the sterileinjectible solutions. Subsequent microbial contamination can beprevented using various antimicrobial agents, for example,antibacterial, antiviral and antifungal agents including parabens,chlorobutanol, phenol, sorbic acid, thiomersal(Ethyl(2-mercaptobenzoato-(2-)-O,S)mercurate(1-) sodium), and the like.Absorption of the desired compounds over a prolonged period can beachieved by including agents for delaying, for example, aluminummonostearate and gelatin.

Nasal spray formulations can include purified aqueous solutions of thedesired compound with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucous membranes. Ophthalmic formulations areprepared by a similar method to the nasal spray, except that the pH andisotonic factors are preferably adjusted to match that of the eye.Formulations for rectal or vaginal administration can be presented as asuppository with a suitable carrier such as cocoa butter, orhydrogenated fats or hydrogenated fatty carboxylic acids.

In addition, a compound of the present disclosure can be modified byappropriate functionalities to enhance selective biological properties.Such modifications are known in the art and include those which increasebiological penetration into a given biological system (e.g., blood,lymphatic system, central nervous system, brain), increase oralavailability, increase solubility to allow administration by injection,alter metabolism and alter rate of exertion. In addition, a compound canbe altered to pro-drug form such that the desired compound is created inthe body of the subject as the result of the action of metabolic orother biochemical processes on the pro-drug. Some examples of pro-drugforms include ketal, acetal, oxime, and hydrazone forms of a compoundthat contains ketone or aldehyde groups.

Preferably, when a compound of the present disclosure can be deliveredin vivo, the dosage level of the compound is on the order of about 10milligrams to about 15 milligrams per kilogram of body weight per day.Preferably, the effective amount is on the order of about 500 milligramsto about 1000 milligrams per subject per day. When a compound of thepresent disclosure is delivered to a subject, the compound can bedelivered in one or multiple dosages for injection, infusion, and/oringestion.

Deuterated compounds according to the present disclosure can havevarious levels of deuterium enrichment. Deuterium enrichment is definedas the percentage of R₁-R_(xx) groups having deuterium atoms. In oneembodiment, the deuterated compound has deuterium enrichment of at least5%. In another embodiment, the deuterated compound has deuteriumenrichment of at least 10%. In another embodiment, the deuteratedcompound has deuterium enrichment of at least 30%. In anotherembodiment, the deuterated compound has deuterium enrichment of at least50%. In another embodiment, the deuterated compound has deuteriumenrichment of at least 70%. In another embodiment, the deuteratedcompound has deuterium enrichment of at least 98%. In anotherembodiment, the deuterated compound has the structure of formula I andhas deuterium enrichment of at least 90%. Determination of the level ofdeuterium enrichment can be performed by mass spectrometry or nuclearmagnetic resonance evaluation.

Examples Synthesis of 7,8-[²H]-3α,7α-dihydroxy-5β-cholan-24-oicacid/7-[²H]-3α,7α-dihydroxy-5β-cholan-24-oic acid (5a,b)

General Methods:

All reagents were purchased from Sigma-Aldrich Corp. (St. Louis, Mo.,USA) and used without further purification unless otherwise noted.Reactions were followed by thin layer chromatography (TLC), carried outusing Merck aluminum backed sheets coated with 60 F254 silica gel, usingEtOAc or EtOAc/n-Hexane mixtures as eluent. Visualization of the TLCspots was achieved by spraying with a 10% solution of sulfuric acid inmethanol followed by heating. Silica gel was acquired from Merck & Co.(White House Station, N.J., USA; 60 G, 0.040-0.063 mm).

¹H- and spectra were recorded on a Bruker Avance III (300 and 100 MHz,respectively). All chemical shifts are quoted on the δ scale in ppmusing residual solvent peaks as the internal standard. Couplingconstants (J) are reported in Hz with the following splittingabbreviations: s=singlet, d=doublet, t=triplet, m=multiplet.

Synthesis of Methyl-3α,7α-dihydroxy-5β-cholanoate (Compound 2)

To a solution of commercial 3α,7α-dihydroxy-5β-cholanic acid (500 mg,1.27 mmol) in MeOH (40 mL) was added p-toluenosulfonic acid and themixture was refluxed under nitrogen atmosphere for 2 hours. The reactionmixture was poured into water (100 mL) and the product was extractedwith EtOAc (3×50 mL). The organics were combined, dried over anhydroussodium sulfate, filtered and concentrated in the rotatory evaporator toyield a colorless oil (516 mg, 1.27 mmol, quantitative yield). Theresidue was pure as judged by TLC and was reacted without furtherpurification.

Synthesis of Methyl-3α-hydroxy-7-oxo-5β-cholanoate (Compound 3)

To a solution of methyl-3α,7α-dihydroxy-5β-cholanoate (Compound 2; 500mg, 1.23 mmol) in CHCl₃ (30 mL, anhydrous) was added silica-gel (2 g)and to this suspension was then added pyridinium chlorochromate (300 mg,1.38 mmol). The reaction was stirred at room temperature for 5 hoursunder nitrogen atmosphere. Et₂O was added (100 mL) and the suspensionwas filtered over a silica-gel column eluting with Et₂O/DCM (7/3) toafford the desired product as a colorless solid (309 mg, 62%). MS(ESI+): m/z 427.1[M+Na]⁺. By-product methyl-3,7-di-oxo-53-cholanoate wasobtained as a colorless solid (154 mg, 31%). MS (ESI+): m/z425.2[M+Na]⁺.

Synthesis of 3α-hydroxy-7-oxo-5β-cholan-24-oic acid (Compound 4)

To a solution of methyl-3α-hydroxy-7-oxo-5β-cholanoate (Compound 3; 269mg, 0.66 mmol) in THF was added LiOH (12 mL, 0.3M aqueous soln) and themixture was stirred at room temperature for 4 hours. The mixture wasacidified with 1M HCl until pH 1 and the product extracted with EtOAc(3×30 mL). The organics were combined, dried over anhydrous sodiumsulfate, filtered and concentrated in the rotatory evaporator to yield acrystalline solid (192 mg, 0.49 mmol, 74%). MS (ESI+): m/z 413.2[M+Na]⁺. ¹H NMR (300 MHz, MeOD) δ 3.60-3.46 (m, 1H), 2.99 (m, 1H), 2.54(t, J=11.2 Hz, 1H), 2.34 (m, 1H), 2.26-2.10 (m, 2H), 2.09-1.71 (m, 7H),1.71-1.25 (m, 9H), 1.25-0.99 (m, 8H), 0.96 (d, J=6.5 Hz, 3H), 0.71 (s,3H).

Synthesis of 7,8-[²H]-3α,7α-dihydroxy-5β-cholan-24-oic acid (Compound5a)/7-[²H]-3α,7α-dihydroxy-5β-cholan-24-oic acid (Compound 5b) (65/35isotopic ratio)

To a refluxing solution of 3α-hydroxy-7-oxo-5β-cholic acid (Compound 4;59 mg, 0.15 mmol) in ^(i)propan(ol-d) (6 mL) in nitrogen atmosphere, wasadded potassium metal (ca. 100 mg) in small pieces and the mixture wasrefluxed for 45 minutes. A 1M HCl soln was carefully added (up to 20 mL)and the product was extracted with EtOAc (3×10 mL). The organics werecombined, dried over anhydrous sodium sulfate, filtered and concentratedin the rotatory evaporator. The product was recrystallized withEtOAc/n-Hexane to yield a colorless solid (Compound 5a,b; 46 mg, 78%).MS (ESI−): m/z 393.0 [M (1D)−1]⁻ (35%); 393.0 [M (2D)−1]⁻ (65%). ¹H NMR(300 MHz, MeOD) δ 3.44-3.57 (m, 1H), 2.15-2.40 (m, 2.4H), 2.02-2.07 (m,1H), 1.79-1.92 (m, 5H), 1.56-1.63 (m, 3H), 1.08-1.53 (m, 15H), 0.87-1.03(m, 5H), 0.72 (s, 3H). Compound 5 is a mixture of Compound 5a(7,8-[²H]-3α,7α-dihydroxy-5β-cholan-24-oic acid) and Compound 5b(7-[²H]-3α,7α-dihydroxy-5β-cholan-24-oic acid).

Cell Culture and Treatments

Primary rat hepatocytes were isolated from male rats (100-150 grams) bycollagenase perfusion (as described by Solá S, et al., 2003, Journal ofBiological Chemistry 278: 48831-48838)). Briefly, rats were anesthetizedwith phenobarbital sodium (100 mg/kg body weight) injected into theperitoneal cavity. After administration of heparin (200 units/kg bodyweight) in the tail vein, the animal's abdomen was opened and the portalvein exposed and cannulated. The liver was then perfused at 37° C. insitu with a calcium-free Hanks' Balanced Salt Solution (HBSS) for about10 minutes, and then with 0.05% collagenase type IV in calcium-presentHBSS for another 10 minutes. Hepatocyte suspensions were obtained bypassing collagenase-digested livers through 125 μm gauze and washingcells in Complete William's E medium (William's E medium, Sigma-AldrichCorp., St Louis, Mo., USA) supplemented with 26 mM sodium bicarbonate,23 mM HEPES, 0.01 units/mL insulin, 2 mM L-glutamine, 10 nMdexamethasone, 100 units/mL penicillin, and 10% heat-inactivated fetalbovine serum (Invitrogen Corp., Carlsbad, Calif., USA). Viable primaryrat hepatocytes were enriched by low-speed centrifugation at 200 g for 3minutes. Cell viability was determined by trypan blue exclusion and wastypically 80-85%. After isolation, hepatocytes were resuspended inComplete William's E medium and plated on Primaria™ tissue culturedishes (BD Biosciences, San Jose, Calif., USA) at 5×10⁴ cells/cm². Cellswere maintained at 37° C. in a humidified atmosphere of 5% CO₂ for 6hours to allow attachment. Plates were then washed with medium to removedead cells and incubated in Complete William's E medium supplementedwith either 100-400 μM UDCA (Sigma-Aldrich Corp.), 100-400 μM deuteratedUDCA (compound 5), or no addition (control). Eight hours afterpre-incubation, cells were exposed to 1 nM recombinant human TGF-β1 (R&DSystems Inc., Minneapolis, Minn., USA) for 36 hours, or to 50 or 100 μMDCA for 16 hours before processing for cell viability, cytotoxicity andapoptosis assays.

Cell Viability, Cytotoxicity, and Caspase Activity Assays

The ApoTox-Glo™ Triplex Assay (Promega Corp., Madison, Wis., USA) wasused to evaluate cell viability, cytotoxicity and caspase-3/7 activity,according to the manufacturer's protocol, using a GloMax+Multi DetectionSystem (Promega Corp.). General cell death was also evaluated using thelactate dehydrogenase (LDH) Cytotoxicity Detection Kit^(PLUS) (RocheDiagnostics GmbH, Mannheim, Germany), following the manufacturer'sinstructions.

Morphologic Evaluation of Apoptosis

Hoechst labeling of cells was used to detect apoptotic nuclei bymorphological analysis. Briefly, culture medium was gently removed toprevent detachment of cells. Attached primary rat hepatocytes were fixedwith 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, for10 minutes at room temperature, washed with PBS, incubated with Hoechstdye 33258 (Sigma-Aldrich Corp.) at 5 μg/mL in PBS for 5 minutes, washedwith PBS, and mounted using Fluoromount-GTM (SouthernBiotech,Birmingham, Ala., USA). Fluorescence was visualized using an Axioskopfluorescence microscope (Carl Zeiss GmbH). Blue-fluorescent nuclei werescored blindly and categorized according to the condensation andstaining characteristics of chromatin. Normal nuclei showednon-condensed chromatin dispersed over the entire nucleus. Apoptoticnuclei were identified by condensed chromatin, contiguous to the nuclearmembrane, as well as by nuclear fragmentation of condensed chromatin.Five random microscopic fields per sample containing approximately 150nuclei were counted, and mean values expressed as the percentage ofapoptotic nuclei.

Statistical Analysis

Statistical analysis was performed using GraphPad InStat version 3.00(GraphPad Software, San Diego, Calif., USA) for the analysis of varianceand Bonferroni's multiple comparison tests. Values of p<0.05 wereconsidered significant.

Results

We tested the cytoprotective and anti-apoptotic effects of newlysynthesized deuterated UDCA (Compound 5), using well-establishedcellular models of apoptosis. It has been previously shown thatnon-deuterated UDCA significantly inhibits DCA-induced apoptosis by ˜60%(Castro et al., 2007, American Journal of Physiology GastrointestinalLiver Physiology 293: G327-G334) and TGF-β1-induced apoptosis by ˜50%(Sold et al., 2003) in primary rat hepatocytes. Cells exposed to 100 and200 μM concentrations of Compound 5 alone showed no relevant signs ofcytotoxicity. Nevertheless, a ˜2% and 15% increase in cytotoxicity wasobserved in cells incubated with the highest concentration of 400 μMCompound 5 for 24 and 44 hours, respectively. This was comparable toUDCA cytotoxicity. In dissecting the cytoprotective effects of Compound5, we tested its ability to prevent DCA-induced cytotoxicity andapoptosis, as compared with UDCA. UDCA and Compound 5 inhibited 50 μMDCA-induced LDH release by 70 and 90%, respectively (FIG. 7A). Thisprotective effect was more than 10% greater for Compound 5 as comparedto UDCA. Slightly reduced protection was obtained for both UDCA andCompound 5 in cells exposed to 100 μM DCA. Notably, Compound 5pretreatment was more effective than UDCA at markedly inhibiting caspaseactivity in cells exposed to both 50 and 100 μM DCA (FIG. 7B). Similarresults were obtained after evaluating nuclear morphology and countingapoptotic nuclei. Nuclear fragmentation induced by 50 μM DCA wasprevented by ˜50% and 65% in cells pretreated with UDCA and Compound 5,respectively (FIG. 7C). Compound 5 was ˜15% more effective than UDCA atreducing the percentage of apoptotic cells. Finally, in cells exposed toa stimulus unrelated to bile acids, Compound 5 was about 10% moreeffective than UDCA at inhibiting TGF-β1-induced LDH release, which inturn was very significantly abrogated (FIG. 8A). In addition, thepercentage of apoptotic cells was reduced by almost 40% after UDCA andCompound 5 pre-treatment (FIG. 8B). Altogether, these results show thatCompound 5 displays powerful cytoprotective and anti-apoptoticproperties in vitro that may even exceed those of UDCA.

FIG. 7 illustrates that Compound 5 prevents DCA-induced cytotoxicity andapoptosis in primary rat hepatocytes. Primary rat hepatocytes wereincubated with 100 μM UDCA, Compound 5, or no addition (control) for 8hours. Cells were then exposed to 50 or 100 μM DCA for 16 hours beforeprocessing for LDH activity (A), caspase-3/7 activity (B) and nuclearfragmentation assays (C). Fluorescent microscopy of Hoechst staining incells exposed to either DCA 50 μM (a), DCA 50 μM+Compound 5 (b), DCA 100μM (c), or DCA 100 μM+Compound 5 (d). Normal nuclei showed non-condensedchromatin dispersed over the entire nucleus. Apoptotic nuclei wereidentified by condensed chromatin, contiguous to the nuclear membrane,as well as nuclear fragmentation of condensed chromatin. Results areexpressed as means±SEM of at least three experiments. *p<0.01 fromcontrol; §p<0.05 and †p<0.01 from respective DCA.

FIG. 8 illustrates that Compound 5 prevents TGF-β1-induced cytotoxicityand apoptosis in primary rat hepatocytes. Primary rat hepatocytes wereincubated with 100 μM UDCA, Compound 5, or no addition (control) for 8hours. Cells were then exposed to 1 nM TGF-β1 for 36 hours beforeprocessing for LDH activity (A), caspase-3/7 activity (B) and nuclearfragmentation assays (C). Fluorescent microscopy of Hoechst staining incells exposed to either TGF-β1 (a) or TGF-β1+Compound 5 (b). Normalnuclei showed non-condensed chromatin dispersed over the entire nucleus.Apoptotic nuclei were identified by condensed chromatin, contiguous tothe nuclear membrane, as well as nuclear fragmentation of condensedchromatin. Results are expressed as means±SEM of at least threeexperiments. *p<0.01 from control; §p<0.05 and †p<0.01 from TGF-β1.

Although the present invention has been described with reference topreferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

1. A deuterated compound selected from the group consisting of:

wherein at least one of elements R₁-R_(XX) comprises deuterium.
 2. Thedeuterated compound of claim 1, wherein the compound is water-soluble.3. The deuterated compound of claim 1, wherein the compound is a salt.4. The deuterated compound of claim 1, wherein the compound ishydrophobic.
 5. The deuterated compound of claim 1, wherein the compoundhas pro-drugs, derivatives and conjugates used for the treatment orprevention of any degenerative disorders in humans.
 6. The deuteratedcompound of claim 1, wherein the compound has deuterium enrichment of atleast 5%.
 7. The deuterated compound of claim 6, wherein the compoundhas deuterium enrichment of at least 10%.
 8. The deuterated compound ofclaim 7, wherein the compound has deuterium enrichment of at least 30%.9. The deuterated compound of claim 8, wherein the compound hasdeuterium enrichment of at least 50%.
 10. The deuterated compound ofclaim 9, wherein the compound has deuterium enrichment of at least 70%.11. The deuterated compound of claim 1, wherein the compound has thestructure of formula I, and wherein the compound has deuteriumenrichment of at least 90%.
 12. The deuterated compound of claim 10,wherein in the compound has deuterium enrichment of at least 98%. 13.The deuterated compound of claim 1, wherein the compound has a structureselected from the group consisting of formulae I, II and III, andwherein the compound comprises one or more PO₄ groups.
 14. Thedeuterated compound of claim 1, wherein the compound has a structureselected from the group consisting of formulae I, II and III, andwherein the compound is chemically conjugated to a pro-drug ofdopaminergic neurons.
 15. The deuterated compound of claim 1, whereinthe compound has a structure selected from the group consisting offormulae I, II and III, and wherein the compound is chemicallyconjugated to a monoamine oxidase inhibitor.
 16. The deuterated compoundof claim 1, wherein the compound has a structure selected from the groupconsisting of formulae I, II and III, and wherein the compound ischemically conjugated to a glutamate receptor antagonists.
 17. Thedeuterated compound of claim 1, wherein the compound has a structureselected from the group consisting of formulae I, II and III, andwherein the compound is chemically modified to function as anantioxidant.